Bone tissue is a heterogeneous material with properties that vary spatially and temporally within the skeleton. Diseases such as osteoporosis affect not only bone quantity but also tissue quality. Therefore, the contribution of tissue composition to bone mechanical properties is being examined in the parent grant (R01-AR053571), with an emphasis on understanding the impact of disease and determining effective treatments. The development and longitudinal growth of long bones occurs through ossification of the cartilage of the growth plate. In contrast to bone and other skeletal tissues, relatively little is known about the mechanical behavior of the growth plate. For this revision we propose to extend our tissue characterization paradigm for bone to the cartilage of the growth plate, a tissue with a unique zonal structure that is strongly linked to its physiologic function. In particular, we will use a technique known as confocal strain mapping to visualize the deformation of the growth plate under load to understand the contribution of this zonal tissue structure to mechanical behavior. In addition, we will use established Fourier Transform Infrared (FTIR) spectroscopy techniques to quantify the zonal distribution of the main structural components of the growth plate, collagen, proteoglycans, and mineral. Together, confocal strain mapping and FTIR spectroscopy will enable us to characterize the relationship between local structure and properties in the growth plate. To accomplish this challenging task, we have assembled an interdisciplinary team of engineers, physical scientists, and biologists with collective expertise in imaging and image analysis, hard and soft tissue characterization, growth plate biology, materials testing and pediatric orthopaedics. The characterization of structure-property relationships in the growth plate will be investigated in two specific aims. Aim 1 will use confocal strain mapping and FTIR spectroscopy to characterize the mechanical behavior and structure of the growth plate of normal growing rats, while Aim 2 will use these same techniques to investigate the effect of dietary vitamin D deficiency on the local mechanical behavior and structure of the growth plate. Vitamin D deficiency is known to have profound affects on growth plate structure, bone mineralization, and, as shown through the parent grant, bone structure and tissue mechanical properties. As such, the work proposed in this revision will give unique insight into the connection between growth plate function and skeletal abnormalities as well as providing a new framework for understanding the physiology of the growth plate and the influence of genetic and environmental factors on this tissue's mechanical performance.